A method for obtaining quantities of clean meloidogyne eggs.

We have obtained gram quantities of clean, viable eggs of Meloidogyne incognita by a method which combined treatment with hypochlorite and differential centrifugation. Chili pepper plants (Capsicum frutescens L.) infected with M. incognita were dug at harvest from a naturally infested field. Egg sacs from roots of 50 plants were obtained by kneading the washed roots in water and recovering the egg sacs on a 60-mesh sieve (2). Egg sacs were placed in an electric blender in 100 ml of tap water, 500 ml of 1% sodium hypochlorite (20% commercial bleach) added, and the blender operated at maximum speed for 40 sec. The resulting suspension of eggs and root debris was poured quickly through nested 100and 400-mesh sieves and eggs which were retained on the 400-mesh sieve were washed with several volumes of distilled water. Eggs which passed through the 400-mesh sieve (pore size 37-/am) were recovered by repeated sieving and rinsing. Eggs were eluted from the sieves, transferred in 40 ml of water to a 50 ml polycarbonate centrifuge tube and centrifuged at 1000 g for 5 min. The pellet, containing eggs and some debris, was resuspended in 40 ml of sucrose solution (454 g sucrose to 1 liter distilled water) and centrifuged at 1000 g for 40 sec. The supernatant was quickly decanted onto a 400-mesh sieve and the eggs retained on the sieve were washed three times with 100 ml portions of distilled water. Eggs that passed through the sieve were recovered by repeating the sieving technique. Dur ing the above procedure, some second-stage larvae were released from the eggs. These larvae were removed by collecting them on a 200-mesh sieve. Eggs were concentrated by centrifugation at 1000 g. By this method, 2 cc (approximately 10 FIG. 1. Eggs ofMeloidogyne incognita. A. Nearly 2 cc of eggs free of debris. B. Photomicrograph of a diluted suspension of debris-free eggs. Bar = 100/am.